The regulation of sodium (Na+) reabsorption across the aldosterone-sensitive distal nephron (ASDN) is critical to the regulation of blood pressure, and known aberrations in this system lead to the onset of hypertension. Serum and glucocorticoid kinase 1, Sgk1, an aldosterone-regulated early-response gene, indirectly stimulates inward Na+ transport via the epithelial Na+ channel, ENaC, by phosphorylating and inhibiting a ubiquitin ligase, Nedd4-2. The mechanism of sgk1 inhibition of Nedd4-2 is unknown and will be the primary focus of this investigation. The hypothesis is that accessory proteins such as 14-3-3 proteins mediate the effects of sgk1 on Nedd4-2 in the distal nephron. Targeted mutants of Nedd4-2 will be constructed and used in a co-expression assay to characterize the role of 14-3-3 proteins in sgk1-mediated inhibition of Nedd4-2. To determine if 14-3-3 proteins physically interact with sgk1-phosphorylated Nedd4-2, a yeast two-hybrid system will be constructed with sgk1-phosphorylated Nedd4-2 as 'bait', and 14-3-3 proteins as 'prey'. Co-immunoprecipitation experiments in a collecting duct cell line will be used to confirm in vivo physical interaction. To study the significance of 14-3-3 proteins on hormone-stimulated Na+ current, RNA interference (RNAi) technology will be utilized to inhibit translation of Nedd4-2, or 14-3-3 proteins in cell culture to determine the effect of each protein on inward Na+ current.